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SIRLEI SAYOMI HAYASHI DETERMINAÇÃO QUALITATIVA E QUANTITATIVA DE METABÓLITOS SECUNDÁRIOS DE Malva sylvestris EM EXTRATOS E ESPECIALIDADES FARMACÊUTICAS Dissertação apresentada ao Programa de Pós- Graduação em Ciências Farmacêuticas, Área de Insumos, Medicamentos e Correlatos, Setor de Ciências da Saúde, Universidade Federal do Paraná, como requisito parcial para a obtenção do título de Mestre. Orientador: Prof. Dr Roberto Pontarolo Co orientadora: Profa. Dra Francinete Ramos Campos CURITIBA 2012 Hayashi, Sirlei Sayomi Determinação qualitativa e quantitativa de metabólitos secundários da Malva sylvestris em extratos e especialidades farmacêuticas / Sirlei Sayomi Hayashi – Curitiba, 2012. 121 f.: il. (algumas color.); 30 cm. Orientador: Professor Dr. Roberto Pontarolo Co-Orientadora: Professora Dra. Francinete Ramos Campos Dissertação (Mestrado) – Programa de Pós-Graduação em Ciências Farmacêuticas, Setor de Ciências da Saúde, Universidade Federal do Paraná. Inclui bibliografia 1. Malva sylvestris. 2. Controle de Qualidade. 3. Fitoterápicos.4. CLAE-DAD. 5. Fingerprint. I. Pontarolo, Roberto. II. Campos, Francinete Ramos. III. Universidade Federal do Paraná. IV. Título. CDD 615.53 AGRADECIMENTOS À agência de fomento à pesquisa CAPES pela concessão da bolsa de estudos. À Universidade Federal do Paraná e ao Programa de Pós graduação de Ciências Farmacêuticas por possibilitar o acesso ao conhecimento. Ao meu orientador Prof. Dr. Roberto Pontarolo pela oportunidade, pelo acolhimento, orientação e incentivo. À minha coorientadora Profa. Dra. Francinete Ramos Campos, pelas incansáveis discussões, pela dedicação com que me orientou e também pelo incentivo. À minha família, pelo apoio, incentivo e torcida. Aos amigos que fiz e aos amigos que me acompanham a tanto tempo que com toda a certeza torceram por este momento. A todos aqueles que diretamente ou indiretamente contribuíram para a realização deste trabalho. Ao Laboratório Herbarium pelo fornecimento dos padrões. A mente que se abre com uma nova idéia, jamais voltará ao seu tamanho original. Albert Einstein RESUMO Malva é o nome polular de Malva sylvestris, planta medicinal utilizada para tratamento de afecções bucais devido a ação antiinflamatória. A fitoterapia atualmente no Brasil vem ganhando força com a implementação da Politica Nacional de Plantas medicinais e Fitoterápicos, de modo que muitas espécies presentes na RENISUS podem ser encontradas nas Unidades de Saúde, entre elas a M. sylvestris. Considerando a nova legislação de registro para medicamentos fitoterápicos, faz-se necessário aprofundar o conhecimento sobre as plantas com utilização terapêutica, bem como definir o metabólito responsável e a quantidade encontrada na espécie, para assegurar a qualidade do fitoterápico. Tendo estas informações definiu-se como objetivo do trabalho desenvolver e validar um método quantitativo para metabólitos da espécie Malva sylvestris por CLAE-DAD, mesmo porque na monografia apresentada pela Farmacopéia Brasileira 4 edição, não consta nenhum método quantitativo para eles. Dentre os metabólitos já relatados na espécie destacam-se os da classe dos flavonóides, cumarinas, compostos fenólicos e antocianidinas. Os metabólitos quantificados foram: ácido cafeico, escopoletina e ácido ferúlico. O método cromatográfico foi definido e validado com coluna C18 XBridge (4,6 x 150 mm; 5 µm), pré coluna C18 XBridge (4,6 x 20 mm; 5 µm), temperatura 25˚C, sistema de fase móvel: Canal A água + ácido fórmico 10% v/v, canal B metanol + ácido fórmico 10% v/v e canal D acetonitrila, fluxo 0,8 mL/minuto e volume de injeção de 5µL. As amostras de extratos secos (M50, M70, M80, M90, M100, S70 e T70) foram pesadas e diluídas em metanol/água 9:1 v/v na concentração de 20 mg/mL de extrato e filtrados em 0,45 µm, enquanto que as amostras comerciais de extratos glicólicos (EC1, EC2, EC3, EC4, EC5, EC6, EC7 e EC8) foram somente filtradas em 0,45 µm. O extrato S70 foi o que apresentou maior concentração de ácido cafeico (17,46 µg/mg), escopoletina (8,09 µg/mg) e ácido ferúlico (15,32 µg/mg). O extrato M50 entre os extratos de maceração foi o que apresentou a maior concentração de escopoletina (4,52 µg/mg). Entre os extratos comerciais que apresentaram maior concentração dos metabólitos foram EC6 e EC7. As analises comparativas dos extratos realizada através das técnicas de CCD, RMN de 1H, CLAE-DAD e CLAE-EM associada a PCA demonstraram que as variações de proporções de etanol e água utilizadas na maceração influenciou na extração de compostos polares, que foi maior nas proporções com maior quantidade de água, enquanto modo de extração avaliados, turbólise seguido de maceração e por fim soxhlet são melhores métodos para compostos polares. O método desenvolvido mostrou-se eficiente para a caracterização e quantificação de três dos metabólitos da M. sylvestris e este pode ser aplicado em análises de controle de qualidade dos extratos comercializados contribuindo para a qualidade dos produtos manipulados e industrializados que contem extrato de malva na sua composição. Palavras chaves: Malva sylvestris. Controle de Qualidade. Fitoterapicos. CLAE- DAD. Fingerprint ABSTRACT Malva is the popular name of Malva sylvestris, herb medicinal used for treatment of oral disease, due to anti-inflammatory action. The growth of phytotherapy in Brazil is due to the implementation of government program called Politica Nacional de Plantas Medicinais e Fitoterápicos. Many species of plants that are available to the population are present in list RENISUS, between them, M. sylvestris. Considering the new legislation of register for herbal medicines in Brazil, becomes necessary to deepen the knowledge about plants with therapeutic use, define this responsible metabolite and the amount found in the species to ensure the quality of the herbal medicine. Therefore, was defined as objective of the work to development and validation of analytical quantitative method for metabolites of M. sylvestris species by HPLC-DAD. Moreover, there is no reported assay method for malva metabolites in Brazilian pharmacopoeia 4th edition monograph. Between the metabolites reported in this species are flavonoid, phenolic compound, coumarins and antocyanidin. The quantified metabolites had been: caffeic acid, scopoletin and ferulic acid. The chromatographic method was defined and validated with column C18 XBridge (4,6 x 150 mm; 5 µm), column guard C18 XBridge (4,6 x 20 mm; 5 µm), temperature 25˚C, system of mobile phase: A - 10% (v/v) formic acid solution, B – methanol/formic acid (90:10, v/v) and D - acetonitrila, at a flow rate 0,8 mL/min gradient elution mode, the detection wavelength was set as 348 nm and the injection volume of 5µL. The dry extract samples (M50, M70, M80, M90, M100, S70 and T70) had been weighed and diluted in (9:1, v/v) methanol/water solution the concentration of 20 mg/mL and filtered in 0,45 µm, and the glycolic extract samples (EC1, EC2, EC3, EC4, EC5, EC6, EC7 and EC8) only had been filtered in 0,45 µm. The S70 extract was what presented greater concentration of caffeic acid (17.46 µg/mg), scopoletin (8.09 µg/mg) and ferulic acid (15.32 µg/mg). The M50 extract presented high er concentration of scopoletin (4.52 µg/mg). Between the commercial extracts, EC6 and EC7 presented greater concentration of the metabolites. Comparative analysis of the extracts was carried through by TLC, RMN of 1H, HPLC-DAD and HPLC-EM associated the PCA. The results had demonstrated that variations of ethanol/water ratio in the maceration affect polar compounds extraction. Higher amounts of water extracted more compounds. Comparing the different methods of extraction turbo extraction was better method followed by maceration and soxhlet. The method developed has provided a efficient for the characterization and quantification of three M. sylvestris’s metabolites. Furthermore, quality control analysis of commercial extracts can apply this method as routine, contributing to quality of medicines products that will countains of malva extract in its composition. Key words: Malva sylvestris. Quality Control. Herbal Medicines. HPLC-DAD. Fingerprint LISTA DE FIGURAS FIGURA 1 - MALVIDINA 3-O-GLICOSÍDEO .............................................................. 25 FIGURA 2 - MALVIDINA 3,5 – DIGLICOSÍDEO ........................................................ 27 FIGURA 3 - QUERCETINA (3,3-4-5,7-PENTAHIDROXIFLAVONA) .......................... 28 FIGURA 4 - KAEMPFEROL (3,4’,5,7-TETRAHIDROXIFLAVONE) ........................... 28 FIGURA 5 – APIGENINA (4’,5,7-TRIHIDOXIFLAVONA) ........................................... 29 FIGURA 6 – GLICINOBETAÍNA (TRIMETILGLICINA)............................................... 30 FIGURA 7 – TRIGONELINA (1 – METILPIRIDINIUM 3 CARBOXILADO) ................. 30 FIGURA 8 – ÁCIDO CAFEICO (ÁCIDO 3-(3,4-DIHIDROXIFENIL-2-PROPENÓICO) .................................................................................................................................. 32 FIGURA 9 – ÁCIDO FERÚLICO (ÁCIDO (E)-3-(4-HIDROXI-3- METOXIFENOL)PROP-2-ENOICO) .......................................................................... 32 FIGURA 10 – ESCOPOLETINA (6-METOXI-7-HIDROXICUMARINA) ..................... 34 FIGURA 11 – ESQUEMA DE MONTAGEM DO SISTEMA DE MACERAÇÃO, MODO CORRETO A, B E C MODO ERRÔNEO D (PRISTA ET AL., 2008) .......................... 35 FIGURA 12 – ESQUEMA DO SOXHLET MODIFICADO FARMACOTÉCNICO ONDE A REPRESENTA O SISTEMA SOXHLET E B A PLACA DE TEFLON UTILIZADA PARA APOIAR A DROGA NO SISTEMA (CARVALHO ET AL., 2009) ......................
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